Ischemic heart disease and heart failure are characterized by the death of large numbers of cardiomyocytes (CMs). The mechanism of death can be due to cellular apoptosis, necrosis or autophagy. The interplay between these processes however remains poorly understood. Moreover tools to image and characterize these three processes simultaneously in vivo have not been developed. The central aim of this proposal is thus to develop a toolbox of molecular imaging probes capable of multiplexed imaging of CM apoptosis, necrosis and autophagy. We hypothesize that the use of this multiplexed approach will provide novel insights into the kinetics and regulation of cell death in the heart. In aim 1, e will develop a library of probes that are suited to downstream in vivo application. Initial validaton will be performed in vitro using flow cytometry of CM cell lines. In aim 2, we will validate the performance of these agents ex vivo in a mouse model of cardiac ischemia reperfusion. The interplay between apoptosis, necrosis and autophagy in the area at risk will be assessed. In addition, the kinetics of cell death in ischemia reperfusion injury will be characterized with this multiplexed approach. In aim 3 continuous wave and lifetime fluorescence imaging, as well as fluorescence tomography, will be performed to image apoptosis, necrosis and autophagy in vivo. In addition, the impact of autophagy will be assessed by multiplexed imaging of transgenic Becn-1 knockout mice. The current proposal extends the prior work of the applicant and his mentors with magnetofluorescent annexins, the gadolinium-decorated vital fluorochrome (Gd-TO) and the in vivo imaging of autophagy with an activatable fluorochrome. Here several modifications will be made to these prior constructs to improve their sensitivity and specificity. The proposal will thus lead to the development of several novel cell death probes, capable, for the first time of multiplexed imaging of apoptosis, necrosis and autophagy in the heart in vivo. In addition, the proposal will provide an important mechanism for the applicant to further his education training and scientific development. The applicant will take courses in bioconjugate chemistry, medical optics, and cardiac pathophysiology at the many universities in the Boston area. He will also receive training in the responsible conduct of research and in biostatistics. The applicant's progress will be monitored frequently by his mentor, co-mentor, and the scientific advisory committee. This NIH Pathway to Independence program will prepare the applicant for his transition to scientific independence, the R00 phase of the application.